Interpretive Summary: Due to the growing preference of consumers for produce grown with a minimum of pesticides, there is a strong need to develop methods of disease control not based on the use of synthetic, chemical fungicides. In recent years, specific species of yeasts have been used as biocontrol agents to manage postharvest diseases of fruits. What makes some species of yeasts more effective as biocontrol agents than other species is relatively unknown. In host-pathogen systems, the recognition of a foreign organism (pathogen) by the host often triggers the production of various forms of oxygen molecules known as reactive oxygen species (ROS). ROS, in turn can trigger an array of plant defense mechanisms or be directly toxic to the invading pathogen. In citrus, we have demonstrated that the ability of a specific postharvest pathogen (Penicillium digitatum) to infect fruit depends on its ability to suppress ROS production by the host. In the current study, we have documented the ability of specific yeast species used as biocontrol agents to produce a burst of ROS when they are placed on the fruit surface. As a result, the fruit itself produces a large amount of hydrogen peroxide. It is suggested that the ability of a yeast species to produce a large amount of ROS when placed on the fruit surface or within a wound and its ability to induce ROS production by host tissues may play a role in its ability to act as a biocontorl agent. Associated with this feature, is the ability of a yeast to tolerate high levels of ROS without experiencing a detrimental effect. This information may be important in the selection of newer, and more effective yeast antagonists to control disease.

Technical Abstract:
The importance of reactive oxygen species (ROS) in plant defense responses against certain pathogens is well documented. There is some evidence that microbial biocontrol agents also induce a transient production of ROS in a host plant which triggers local and systemic defense responses to pathogens. The ability of biocontrol agents used to control postharvest diseases to induce defense-related oxidative responses in fruits, however, has not been explored. Here we show that antagonist yeast, Metschnikowia fructicola and Candida oleophila generate greater levels of super oxide anion on intact fruit surfaces (poor in nutrients) then those applied on a nutrient-poor agar medium. Even though antagonistic yeast show an increased production of super oxide anion on nutrient-rich media, when applied on fruits around wounds, accumulation of super oxide anion, as detected by nitro blue tetrazolium staining, occurred much more rapidly on the latter. Using laser scanning confocal microscopy we observed that the application of M. fructicola and Candida oleophila into citrus and apple fruit wounds correlated with an increase in H(2)O(2) accumulation in host tissue. As early as 18 hours after inoculation, the level of H(2)O(2) around inoculated wounds increased by 4-fold compared to controls (wounds inoculated with water). Yeast continued to stimulate H(2)O(2) production in citrus fruit up to 66 h after inoculation and H(2)O(2) levels were still threefold above the control. Living yeast cells were detected in fruit wounds at this time point indicating the ability of M. fructicola to tolerate host ROS, which has been reported to be an intrinsic characteristic of efficient yeast antagonists. The present data, together with our earlier discovery of the importance of H(2)O(2) production in the defense response of citrus flavedo to postharvest pathogens, indicate that the ability of yeasts to induce an oxidative response in fruit tissue may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases.